Fine-needle aspiration biopsy and flow cytometry immunophenotyping of lymphoid and myeloproliferative disorders of the spleen

Fine-Needle Aspiration Biopsy and Flow CytometryImmunophenotyping of Lymphoid andMyeloproliferative Disorders of the Spleen

Pio Zeppa, M.D.1

Marco Picardi, M.D.2

Gilda Marino, M.D.1

Giancarlo Troncone, M.D.1

Franco Fulciniti, M.D.1

Antonio Vetrani, M.D.1

Bruno Rotoli, M.D.2

Lucio Palombini, M.D.1

1 Dipartimento di Anatomia Patologica e Citopato-logia, Facolta di Medicina e Chirurgia, Universita diNapoli “Federico II,” Napoli, Italy.

2 Dipartimento di Medicina Interna, Area di Ema-tologia, Facolta di Medicina e Chirurgia, Universitadi Napoli “Federico II,” Napoli, Italy.

The authors thank Dr. Antonino Iaccarino and Dr.Roberto Arrichiello for technical assistance andMrs. Mary Spears for reviewing the manuscript.

Address for reprints: Lucio Palombini, M.D., Dipar-timento di Anatomia Patologica, Facolta di Medi-cina e Chirurgia, Universita di Napoli “Federico II,”Via Pansini 5, 80131 Napoli, Italy; Fax: 011-39-081-7463679; E-mail: [email protected]

Received May 18, 2002; revision received August15, 2002; accepted September 3, 2002.

BACKGROUND. Flow cytometry (FC) is a useful adjunct to fine-needle aspiration

biopsy (FNAB) in the evaluation of lymphoproliferative disorders. The application

of FC to FNAB of the spleen (sFNAB) is reported.

METHODS. Flow cytometry was performed on 18 sFNAB collected over 3 years. The

series comprised 10 cases of non- Hodgkin lymphomas (NHL), 2 cases insufficient

for diagnosis, 2 cases of reactive hyperplasia (RH), and 4 cases of myeloid meta-

plasia (MM). FNAB was performed under ultrasound guidance using a 22-gauge

needle. One or two passes were sufficient to prepare a conventional smear that was

immediately evaluated to select the cases studied and to prepare a cell suspension

for FC. The following fluoresceinated antibodies were used: CD3, CD19/�/�,

FMC7/CD23/CD19, Bcl-2, and CD13/HLA-DR. In six cases, cytospins were also

prepared for immunocytochemistry and were tested for CD20 (L26), CD45Ro, and

� and � light chain expression.

RESULTS. Flow cytometry contributed to the diagnosis of all cases of NHL by

assessing light chain restriction. The specific subtype was also diagnosed by

CD19/CD5 and CD 19/CD10 coexpression in two cases. Flow cytometry quantified

the percentage of myeloid cells in MM cases and contributed to the cytologic

diagnosis showing a polyclonal light chain expression in RH cases.. Immunocyto-

chemistry was effective and concordant in four cases. Patients tolerated the sFNAB

well and no complications were reported. Cytologic and FC diagnoses were con-

firmed by follow-up and by histologic evaluation in cases in which splenectomy

was performed for therapeutic purposes.

CONCLUSION. Flow cytometry applied to sFNAB corroborates the cytologic diag-

nosis in lymphoid and myeloproliferative disorders of the spleen and allows

therapeutic decisions avoiding splenectomy. Cancer (Cancer Cytopathol)

2003;99:118 –27. © 2003 American Cancer Society.

KEYWORDS: fine-needle aspiration biopsy, spleen, flow cytometry, immunopheno-typing.

F ine-needle aspiration biopsy of the spleen (sFNAB) was first usedin 1916 for the diagnosis of leishmaniasis.1 In 1970, Soderstrom2

used the technique extensively to diagnose splenomegaly. Because ofthe general skepticism toward the cytologic diagnosis of lymphopro-liferative disorders and the fear of hemorrhage or rupture, the appli-cation has not been reported much3–21 and has not become as widelyused as in other organs. In addition, the tremendous progress ofnoninvasive diagnostic tools and imaging techniques has reduced theneed of direct investigation of this organ through biopsies or sple-nectomy. However, there are clinical situations, mainly concerninglymphoid and myeloproliferative disorders, in which clinical and

118 CANCERCYTOPATHOLOGY

© 2003 American Cancer Society

DOI 10.1002/cncr.11011

instrumental information is not sufficient to diagnosesplenic nodules or diffuse splenomegalies. Splenec-tomy for diagnostic purposes can sometimes be “toomuch” or useless in the management of lymphoid andmyeloproliferative disorders and sFNAB should beused to obtain a correct diagnosis. In recent years,flow cytometry (FC) has proven to be useful in theevaluation of lymphoproliferative disorders usingspecimens of lymph nodes obtained from surgicalprocedures or from FNAB.22–33 A few reports havedealt with splenic lesions on cytologic or surgical sam-ples.23,34,35

We report our experience with the application ofFC to sFNAB and evaluate the possible contribution ofthe combined techniques to the diagnosis of splenicnodule or splenomegaly.

MATERIALS AND METHODSFlow cytometry was used to analyze 18 sFNAB per-formed between January 1999 and December 2001.The sFNAB were performed using ultrasound (US)guidance in the outpatient clinic of the HematologyDepartment on patients with (nine cases) or without(nine cases) history of lymphomas or other hemato-logic disorders. Clinicopathologic data of all cases aresummarized in Table 1. At the time of the sFNAB, thediagnostic procedure and its related risks were dis-cussed with the patients and informed consent wasobtained, after which a preliminary clinical and USexamination was performed. According to Kline,36

hemorrhagic diathesis and infective mononucleosiswere absolute contraindications.

The sFNAB were performed under US guidanceusing an EUB 525 Hitachi instrument (Tokyo, Japan)and a 3.5-MHz curvilinear probe (connected to apuncture adaptor during the invasive procedure),guide, and a 22-gauge modified Chiba needle, 150 mmin length (Ecojekt, Hospital Service, Rome, Italy). Weverified access to the organ, generally preferring thesubcostal approach, and measured the distance be-tween the skin and the target. The guide has a definedangle that does not change when the probe is moved.Therefore, the needle can be followed along the passuntil the tip reaches the center of the nodule or theparenchyma in case of diffuse splenomegalies. At themoment of the maneuver, patients were instructed tohold their breath.

The first pass was used to prepare two traditionalsmears. The first smear was stained immediately withDiff-Quik (American Scientific Products, McGrawPark, IL) and evaluated for suitability for immunophe-notyping. The other smear was fixed in alcohol for Papstaining. The remaining material in the hub of theneedle was flushed carefully with phosphate-buffered

saline solution (PBS), which was added to an eventualsecond pass in cases of scanty cellularity. Two sFNABsmears had insufficient cellular material because ofabundant necrosis and blood, respectively. In bothcases, a cytologic diagnosis was not possible and FCwas not performed. One patient had metastatic dis-ease and that smear was not included in the currentstudy. An US scan was then repeated to role out anyimmediate complication and patients were advised torest in bed for few hours with ice packs on the splenicarea.

ImmunocytochemistrySix cases yielded enough material to prepare four cy-tospins each, which were used for immunocytochem-ical stains. They were tested for CD20 (L26), CD45 RO(pan T), and � and � light chains with a three-stepimmunoperoxidase technique.19 The sources of theprimary antibodies and dilutions are summarized inTable 2.

FCThe sFNAB suspensions were sent immediately to thelaboratory of the Cytopathology Service (Departmentof Pathology, University of Naples) where they werewashed twice by centrifugation for 5 minutes at 2500rpm. The supernatant was removed and 400 �L of PBSwas added. The final suspension was divided into fouror more tubes when sufficient cells were available.

Samples were incubated for 15 minutes in thedark with 10 �L of the following basic combinations ofphycoerythrin, peridinin chlorophyll protein, and flu-orescein isothiocyanate antibodies: CD3, CD5/10/19,CD19/�/�, and FMC7/CD23/CD19. These combina-tions were used as a basic panel in all cases accordingto the experiences of others.33,37 The basic panel re-quires four tubes. However, for six cases, we dividedthe suspension among five tubes on the basis of clin-ical data and cytologic features. The fifth tube wasused to test for bcl-2 or CD13-HLA-DR. When diag-nostic material was sufficient, immunophenotypingwas performed on cytospins. All antibodies were pur-chased from Becton Dickinson (San Jose, CA), exceptbcl-2, which was purchased from Pharmingen (SanJose, CA). Table 2 lists all antibodies and correspond-ing conjugated fluorochromes.

After incubation, red blood cells were lysed withammonium chloride lysing solution (diluted to 10%)for 15 minutes and washed. When small fragmentswere still present, the suspension was filteredthrough 50-�m filters. Finally, the samples wereacquired until 10,000 events were analyzed for eachtube. Flow cytometry was performed using a three-color analysis technique on a Becton Dickinson

Flow Cytometry in FNAB of the Spleen/Zeppa et al. 119

FACScan. Flow cytometric evaluation of the datawas performed with a doublet discrimination mod-ule. After acquisition, the lymphoid cell populationwas selected arbitrarily from gating events between200 and 600. A minimum of 1000 gated events wasconsidered adequate.

Data analysis was performed using PAINT-A-GATE PRO software (Becton Dickinson). The pheno-type of the gated cell populations was determined bythe distribution of the painted cell population in mul-

tiparameter space. Events proximal to the axis chosenfor the specific fluorochrome were considered to bespecific for the corresponding conjugated antibodyand double-marked events were localized in the upperright quadrant equidistant from both the axes. As tothe light chain evaluation, �-to-� and �-to-� ratiosgreater than 6:1 and 4:1, respectively, were definiteevidence of monoclonality.22 Flow cytometric datawere reviewed with the cytologic features and immu-nocytochemical data.

TABLE 1Clinical, Cytologic, Cytofluorometric, and Immunocytochemical Data of 18 Fine-Needle Aspiration Biopsies of the Spleen

Caseno. Gender/age Clinical data

Ultrasoundfeatures Immunocytochemistry Flow cytometry

Cytologicdiagnosis

Histologicdiagnosis

1 M/81 Negative Diffusesplenomegaly

Not performed CD3�, CD5�, CD10�, CD19�, CD23�,FMC7�, CD13�, HLA-DR�, K�, ��

Myeloidmetaplasia

Not performed

2 F/44 Follicular NHLinremission

Multiple smallnodules

Not performed CD3�, CD5�, CD10�, CD19�, CD10/19�, CD23�, FMC7�, Bcl-2�, K�,��

Follicularmedium-cell NHL

Not performed

3 M/34 Negative Diffusesplenomegaly

Not performed CD3�, CD5�, CD10�, CD19�, CD23�,FMC7�, Bcl-2�, K�, ��

Reactivehyperplasia

Not performed

4 F/61 Negative Single noduleand lymphnodes

CD45Ro�, CD20�, K�,��

CD3�, CD5�, CD10�, CD19�, CD23�,FMC7�, K�, ��

Large B- cellNHL

Not performed

5 M/81 Myelofibrosis Diffusesplenomegaly


Myeloidmetaplasia

Not performed

6 F/59 Negative Single nodule CD45Ro�, CD20�, K�,��

CD3�, CD5�, CD10�, CD19�, CD23�,FMC7�, K�, ��

Large B-cellNHL

Large B-cell NHL

7 M/66 Follicular NHLinremission


Not performed Not performed Insufficient Performed on alymph node:large B-cellNHL

8 F/55 NHL inremission

Diffusesplenomegaly

Not performed CD3�, CD5�, C010�, CD19�, CD23�,FMC7�, K�, ��

Medium B-cell NHL

Not performed

9 F/48 Negative Single nodule Not contributive CD3�, CD5�, CD10�, CD19�, CD23�,FMC7�, K�, ��

Large B- cellNHL

Large B-cell NHL

10 F/70 Negative Single nodule Not performed CD3�, CD5�, CD10�, CD19�, CD23�,FMC7�, K�, ��

Large B- cellNHL

Large B-cell NHL

11 M/70 NHL inremission

Diffusesplenomegaly

CD45Ro�,CD20�, K�, ��

CD3�, CD5�, CD10�, CD19�, CD23�,FMC7�, K�, ��

Medium B-cell NHL

Not performed

12 F/60 Negative Single nodule Not performed CD3�, CD5�, CD10�, CD19�, CD5/CD19�, CD23�, FMC7�,CD19/FMC7�, K�, ��

Mantle cell,mediumB-cellNHL

Mantle cell NHL

13 M/68 NHL inremission

Diffusesplenomegaly

Not performed CD5�, CD10�, CD19�, CD23�,FMC7�, K�, ��, K/��6:1

Medium B-cell NHL

Not performed

14 F/55 Negative Diffusesplenomegaly

CD20�,CD45Ro�, K�, ��

CD3�, CD5�, CD10�, CD19�, CD23�,FMC7�, Bcl2�, K�, ��

Reactivehyperplasia

Not performed

15 F/52 Myelofibrosis Diffusesplenomegaly


Myeloidmetaplasia

Not performed

16 F/72 Negative Single nodule Not performed Not performed Insufficient Large B-cell NHLwith necrosis

17 M/51 Peripheraleosinophilia



Myeloidmetaplasia

Not performed

18 F/48 NHL inremission

Diffusesplenomegaly

Not contributive CD3�, CD5�, CD10�, CD19�, CD23�,FMC7�, Bcl2�, K�, ��

Medium-B-cell NHL

Not performed

NHL: non-Hodgkin lymphoma.

120 CANCER (CANCER CYTOPATHOLOGY) April 25, 2003 / Volume 99 / Number 2

RESULTSThe current series comprises 14 cases of non-Hodgkinlymphoma (NHL), 4 cases of myeloid metaplasia(MM), and 2 cases of benign white pulp hyperplasias.Six cases of primary NHL of the spleen were revealedat the US examination by the presence of large nod-ules, and, in one case, lomboaortic lymph node in-volvement was discovered at the time of the sFNAB.The other seven cases concerned patients with a pre-vious history of NHL in remission who developed dif-fuse splenomegaly (five cases) or multiple small (mili-aric) splenic nodules (two cases). Two insufficientcases included multiple small nodules or hemorrhageat the aspiration site (Case 7) and nodular NHL withextensive necrosis (Case 16). In these cases, two passesdid not obtain sufficient material. Additional passeswere not performed because of the necrosis and thefear of hemorrhage. Therefore, this patient (Case 16)underwent splenectomy. In the other patient (Case 7),a lomboaortic lymph node was discovered at the USexamination and final diagnosis was performed onthis lymph node.

In four cases of primitive NHL with single-nodulepresentation, including one case with lomboaorticlymph nodes (Cases 4, 6, 9, 10), the cytologic presen-tation showed a relative monomorphous proliferationof immature large lymphoid cells with irregularlyshaped, dispersed, and coarse chromatin and evidentnucleoli (Fig. 1A). These cases were classified as large-cell, high-grade NHL. In Case 12, which had single-nodule presentation, the smear showed medium, dis-persed lymphoid cells with an irregularly shapednuclear membrane and small nucleoli (Fig. 2A). Thiscase was classified as medium-cell NHL. In five cases(Cases 2, 8, 11, 13, 18) with diffuse or multinodularpresentation, smears showed medium cells withclumped chromatin and, if present, small nucleoli. No

dense fragments of lymphoid cells with a vascularcore, i.e., periarteriolar lymphoid sheets (PALS),19,37

were observed. In all these cases, FC showed � or �

light chain restriction and a prevalence of CD19 cells.In Case 2, the coexpression of CD10/CD19 identified afollicular lymphoma. In Case 12, the coexpression ofCD5/CD19 (Fig. 2B), CD19/FMC7 (Fig. 2C), and � lightchain restriction (Fig. 2D) identified a mantle cell lym-phoma. In all the other cases, CD19 prevalence andlight chain restriction together with the cytologic fea-tures allowed a diagnosis of B small or large-cell NHLwithout any other subclassification. Two patients(Cases 2 and 18) with a history of follicular NHL testednegative for bcl-2. No T-cell lymphomas were ob-served.

Four patients in the current study were diagnosedwith MM. Their US scan revealed diffuse splenomeg-aly (Fig. 3A). Two had a history of myelofibrosis, onehad peripheral eosinophilia, and one did not have ahistory of MM. The microscopic pattern showedmegakaryocytes and myeloid cells at different stagesof maturation entrapped in small fragments of whitepulp (PALS) or spread in the background (Fig. 3B).Flow cytometry showed a small population of CD19and HLA-DR–positive cells with a balanced ratio of thelight chains and a second population of CD13-positivecells (Fig. 3C). Two cases of white pulp hyperplasiapresented with diffuse splenomegaly. The smearsshowed lymphoid cells in different stages of matura-tion, conferring a polymorphous appearance, andsmall dense fragments with a central vascular core(PALS; Fig. 4A). Flow cytometry showed two CD19 andCD5-positive populations (Fig. 4B) and a balanced�-to-� light chain ratio (Fig. 4C). The results of theBcl-2 test were negative. Immunocytochemistry didnot contribute to the diagnosis in two cases because ofinsufficient material in the cytospins. However, it sup-

TABLE 2Primary Antibodies Used for ICH and FC

Antibody Technique Source Dilution Incubation

CD 20 (L26) ICH Menarini (Firenze, Italy) 1:100 30 min RTCD45Ro (pan T) ICH Menarini (Firenze, Italy) 1:100 30 min RTKappa light chain ICH Dako (Dakopatts, Glostrup, Denmark) 1:20,000 30 min RTLambda light chain ICH Dako 1:20,000 30 min RTCD3(PE) FC Becton Dickinson (San Jose, CA) 1 �L: 100a 15 min in the dark, RTCD5 (FITC)/CD10 (PE)/CD19 (PerCp) FC Becton Dickinson 1 �L: 100a 15 min in the dark, RTFMC7 (FITC)/CD23 (PE)/CD19 (PerCp) FC Becton Dickinson 1 �L: 100a 15 min in the dark, RTCD19 (FITC)/K (PE)/� (PerCp) FC Becton Dickinson 1 �L: 100a 3 min in the dark, 4°CCD13 (PE)/HLA-DR (FITC) FC Becton Dickinson 1 �L: 100a 3 min in the dark, 4°CBcl-2 (FITC) FC Pharmigen (San Jose, CA) 1 �L: 100a 3 min in the dark, 4°C

ICH: immunocytochemistry; FC: flow cytometry; PE: phycoerythrin; PERCP: peridinin chlorophyll protein; FITC: fluorescein isothiocyanate; RT: room temprature.a One milliliters of cell suspension.


ported the diagnosis of NHL in four cases. In one case,scattered cells stained positively for B and T-cell lin-eage markers and � and � light chains. In the otherthree cases, immunostaining was positive for pan Bwith � light chain restriction and negative for pan-Tand � light chains, demonstrating the monoclonalityof the cell population and supporting the cytologicand FC diagnosis of NHL.

Five patients with primary NHL presenting aslarge splenic nodules (sNHL) underwent splenectomy,as did one patient who had an inconclusive sFNAB.The subsequent histologic evaluation confirmed thecytologic-cytofluorometric diagnosis of large B-cellNHL with extensive necrosis. Therapeutic procedures

were performed without histologic control in all pos-itive cases with a previous story of NHL, as well as inone case (Case 4) because of the concordance of clin-ical presentation, cytologic diagnosis, and FC. In neg-ative cases, a thorough clinical follow-up confirmedthe cytologic diagnosis. In fact, patients with MM andbenign hyperplasia underwent a clinical follow-upand US examination. Splenomegaly in two patientswith benign reactive hyperplasia was discovered dur-ing a general check-up and during clinical investiga-tion of an idiopathic fever, respectively. No other sig-nificant enlargement of lymph nodes or other organswas found. Both patients are alive 2 years later. Twopatients with MM suffered from myelofibrosis at the

FIGURE 1. (A) Ultrasound imaging of non-Hodgkin lymphoma presenting as a single splenic nodule. Note the tip of the needle. (B) Cytologic features show atypical

nucleolated large cells (Diff-Quik stain, � 430). (C) Flow cytometry shows large cluster of CD19-positive (upper left quadrant) and small CD5-positive cells (lower

right quadrant). (D) Flow cytometry shows monoclonal population of B cells showing � light chain restriction (lower right quadrant).


time of sFNAB. Two patients without a history of MMwere shown to have myelofibrosis after a hematologicscreening, including a bone marrow examination. Af-ter 2 years, all these patients are alive with disease.Clinical, cytologic, and immunocytochemical and cyt-ofluorometric data are summarized in Table 1.

DISCUSSIONFine-needle aspiration biopsy is not used routinely todiagnose lymphoproliferative disorders because it is

not informative of the architecture, which is a limita-tion to the diagnostic interpretation of the smears.37–39

Ancillary techniques such as immunocytochemistryand FC have gained more importance in the diagnosisof lymphoproliferative disorders. The last revised Eu-ropean-American Classification of lymphomas41 en-hanced the immunophenotyping and the cytologicfeatures of lymphoid populations more than the con-ventional nodular or diffuse growth pattern, which hastraditionally characterized other classification systems

FIGURE 2. (A) Cytologic features of a nodular mantle cell non-Hodgkin lymphoma . Medium cells show frequent irregularly shaped nuclei (Diff-Quik stain, � 430).

(B) Flow cytometry shows coexpression of CD19/CD5 (upper right quadrant). (C) Flow cytometry shows coexpression of CD19/FMC7 (upper right quadrant). (D) Flow

cytometry shows � light chain restriction (upper left).


of malignant lymphomas. Consequently, FNAB hasgained credibility, mainly when a contextual immuno-phenotyping of the cell population is performed.39,40

Until recently, we used immunocytochemistry oncytospin samples of lymphoproliferative processes toassess the phenotype of aspirated cells. In our experi-ence, scanty cellularity and technical difficulties werethe disadvantages of this method. These difficulties,which were encountered with lymph node samples,were even greater in splenic samples, which generallyare scanty and hemorrhagic. Flow cytometry has beenused successfully to evaluate lymph nodes on FNABsmears from patients with lymphoproliferative disor-ders. Three years ago, we began to perform immuno-phenotyping on sFNAB by FC.

A basic panel uses four tubes to assess the clonal-ity of the cell population and to distinguish among

various types of low-grade NHL (follicular lymphoma,benign reactive hyperplasia, MM).33–38,40 In the cur-rent study, we used five tubes as discussed in theMaterials and Methods. When diagnostic material wassufficient, immunophenotyping was performed on cy-tospin samples to compare the FC results. We diag-nosed five cases of primary splenic NHL, one splenicinvolvement by NHL with concomitant positive lom-boaortic lymph nodes, six cases of NHL during thefollow-up of patients with existing disease, four casesof MM, and two cases of benign lymphoid hyperplasia.

We observed that the absolute incidence of thedifferent pathologic entities in our series and of thesubtypes of NHL was different when compared withthe real incidence of the same entities in thespleen.40,42 Our cases of primary sNHL, splenic in-volvement during NHL without lymph node presenta-

FIGURE 3. (A) Ultrasound imaging of myeloid metaplasia presenting as

diffuse splenomegaly. Note the tip of the needle. (B) Cytologic features show

myeloid and megakaryocytic cells interspersed in the background and a

dense lymphoid fragment (Diff-Quik stain, � 430). (C) Flow cytometry shows

CD13-positive cells (myeloid; upper left quadrant) and HLA-DR–positive cells

(lymphoid; lower right quadrant).


tion or other involved organs, and MM with severesplenomegaly were less represented in other se-ries.40,42 In all the sNHL cases in our series, a definitelight chain restriction was demonstrated, which wasessential for diagnosis of low-grade NHL and benignwhite pulp hyperplasia. In lymph node or extra lymphnode lymphoproliferative processes, light chain re-strictions should be evaluated carefully in cases withfew gated cells.22

In the current study, there were more than 1000gated cells for light chain evaluation of all cases.Therefore, we were confident that all of our cases weremonoclonal. The evaluation of the FC panel, com-bined with the cytologic features, allowed the correctsubclassification of two cases that were identified asfollicular by the coexpression of CD19/CD10 andmantle cell by the coexpression of CD19/CD5 andCD19/FMC7. In all other cases, sNHL with light chain

restriction was diagnosed without subclassification.The percentage of subclassifications in the currentstudy series is low compared with other series.33 Webelieve that this is due to the high percentage of large-cell NHL in the current study, which represents agenetically heterogeneous group of NHL not carryinga single phenotype. It has been proven that FC ishighly sensitive in the subclassification of low-gradeNHL (e.g., small lymphocytic, follicular, and mantlecell NHL) and is less specific with the high-grade tu-mors.33

As the current cases are mainly primary sNHLwith one or more large splenic nodules, the incidenceof large cell types among these cases might explain therelative low rate of subclassification. The differentialdiagnosis of the two cases of white pulp hyperplasiawith a low-grade and/or follicular center sNHL wasmade considering the relative polymorphism of the

FIGURE 4. (A) Cytologic features of white pulp reactive hyperplasia.

Polymorphous lymphoid cells are interspersed among small dense lymphoid

fragments (Diff-Quik stain, � 430). (B) Flow cytometry shows two cell

populations, namely, CD5 and CD19-positive cells. (C) Flow cytometry ex-

pression of both � and � light chains.


cell population and the presence of PALS that sug-gested a reactive hyperplasia. Cytologic diagnosis wasconfirmed by FC through the demonstration of poly-clonality of the light chains and the negativity forbcl-2. One of these cases (Case 3) was CD10 positiveand showed a slight CD10/CD19 coexpression. Thisphenotype is generally representative of a follicularhyperplasia and should not be considered an NHLunless there is a light chain restriction.33 Myeloidmetaplasia was observed in four cases with myelofi-brosis (Cases 5, 14), peripheral eosinophilia (Case 15),or idiopathic fever (Case 1). As myelofibrosis is a my-eloproliferative disorder, sFNAB was required to ruleout a possible extramedullary leukemia and, in thefourth case, to investigate an unexplained splenomeg-aly. As reported elsewhere,19,40 identification ofmegakaryocytes is the hallmark of this diagnosis. Cy-tologic features and FC ruled out leukemia. In addi-tion, FC quantified the rate of metaplastic myeloidcells by gating the CD13 (mature myeloid) andHLA-DR (lymphoid) events.

In conclusion, sFNAB was well tolerated by allpatients and no complications occurred. FC applied tosFNAB material provided a definite diagnosis in lym-phoid and myeloproliferative disorders, enhancing thediagnostic power of the sFNAB.

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Fine-needle aspiration biopsy and flow cytometry immunophenotyping of lymphoid and myeloproliferative disorders of the spleen